Single-Cell Recombinant Pharmacology: Bovine α1a-Adrenoceptors in Rat-1 Fibroblasts Release Intracellular Ca2+, Display Subtype-Characteristic Agonism and Antagonism, and Exhibit an Antagonist-Reversible Inverse Concentration-Response Phase11This work was supported by Medical Research Council, European Commission, Pfizer, and the British Heart Foundation.

Phe-activated Ca2+signals recorded from single rat-1 fibroblasts stably expressing the bovine α1a-adrenoceptor (AR) were characterized and used to analyze functional agonist-antagonist interactions. The response to Phe was initiated by the mobilization of stored Ca2+and subsequently sustained by receptor-regulated Ca2+influx. The selective α1A-AR agonist (R)-A-61603 was 141-fold more potent as an agonist than Phe. This potency ratio was consistent with the pharmacology of the native α1A-ARs. Functional responses evoked by concentrations of Phe of more than 0.3 μM displayed fade, which could be explained by agonist-dependent depletion of Ca2+stores. The antagonists tested did not conform to the predictions of the Schild equation for competitive antagonism as expected from the nonequilibrium nature of the response. The antagonist potency series WB4101 ≥ prazosin ≫ BMY7378, however, was consistent with α1A-ARs. Antagonism exhibited by WB4101 and prazosin was compatible with a model in which antagonists dissociate so slowly from the receptor that this is a major factor in their inhibition of the transient agonist-mediated response, leading to the appearance of insurmountable antagonism. A consequence of this phenomenon was that an inverse concentration-response relationship at high agonist concentrations was abolished by low concentrations of antagonists. Overall, the results indicate that quantitative pharmacology can be studied successfully in single cells even though equilibrium could not be achieved in the agonist-antagonist-response relationship in this particular cell phenotype. The study also showed a form of fade that could be readily explained.